Anti-apoptotic and neuroprotective erythropoietin/CRLF3-signalling in insects and humans

The cytokine receptor like factor 3 (CRLF3) evolved together with the eumetazoan nervous system and is present in all major groups of Animalia. Based on sequence similarities, CRLF3 was assigned to the family of class 1 cytokine receptors which also includes the classical erythropoietin receptor (EpoR). CRLF3 misregulation has been associated with several human diseases, but neither its ligand nor a particular function have been reported. Erythropoietin (Epo) is a vertebrate-specific helical cytokine that regulates erythropoiesis and activates cytoprotective pathways in various tissues including the nervous system. Neuroprotective functions of Epo are partially mediated by EpoR but also by additional, partly unidentified receptors. In insects, CRLF3 was identified to respond to human recombinant Epo and the naturally occurring Epo splice variant EV-3. Activation of CRLF3 in insects stimulates anti-apoptotic processes via JAK/STAT intracellular signalling. Even though many efforts were made in order to characterize CRLF3-mediated responses, downstream effectors beyond JAK/STAT remained elusive. My thesis combines studies on CRLF3-mediated anti-apoptotic mechanisms in insects and the functional characterisation of human CRLF3 in iPSC (induced pluripotent stem cell) -derived neurons. Studies on Locusta migratoria and Tribolium castaneum revealed a pro-apoptotic role of acetylcholinesterase (AChE coded by ace) that was previously reported for vertebrates. Similar to those studies, reduction of AChE levels and inhibition of AChE activity prevented apoptotic death in hypoxiaexposed primary neuron cultures. Moreover, apoptogenic stimuli increased ace expression supporting the association of AChE with increased apoptosis under challenging conditions. Experiments in T. castaneum indicated that both types of AChE (AChE-1 transcribed from ace-1 and AChE-2 transcribed from ace-2) promote apoptosis and are upregulated by apoptogenic stimuli. However, stressinduced upregulation of AChE-1 was prevented by neuroprotective concentrations of Epo. This indicated that Epo/CRLF3-stimulated neuroprotection is mediated through suppression of pro-apoptotic ace-1 expression. Whether this Epo-stimulated protective mechanism is specific to insects or also present in other species remains to be studied. In order to determine the endogenous ligand of insect CRLF3 (insects do not possess Epo) I used locust hemolymph as potential source for its identification. I first demonstrated that locust hemolymph protects both L. migratoria and T. castaneum primary neurons from hypoxia-induced apoptosis. The protective effect was absent after RNAi-mediated knockdown of CRLF3 expression. Thus, locust hemolymph contains a ligand that is sufficiently conserved to activate CRLF3 in different insect species. Fractionation of locust hemolymph by size exclusion chromatography generated two (out of >11) fractions with particular neuroprotective potency. These hemolymph fractions will be used to separate and identify the CRLF3 ligand. In order to determine the function of human CRLF3, I generated CRLF3-knockout lines from two fibroblast-derived human iPSC lines by CRISPR/Cas9 gene editing. CRLF3 KO lines, along with wild type and isogenic controls, were differentiated into neuronal-like cells expressing cell type-specific markes and presenting characteristic morphology. After differentiation, neuronal-like cells were exposed to rotenone, an inhibitor of respiratory chain complex I, which induced apoptosis in all cell lines. The addition of the Epo splice variant EV-3 prevented rotenone-induced cell death in wild type and isogenic controls but not in CRLF3 KO neurons. This demonstrates that human CRLF3 is a neuroprotective receptor similar to its previously determined function in insects. Moreover, CRLF3 is identified as the first known receptor for EV-3 and vice versa, human CRLF3 is deorphanized by identifying EV-3 as one of its endogenous ligands. Taken together, my work has identifyed CRLF3 as a new player in neuroprotection that may account for various previously described Epo-mediated cytoprotective functions in the nervous system and other tissues. Specific activation of CRLF3-mediated beneficial pathways may interfere with degenerative processes without coactivation of EpoR and its associated adverse side effects.2023-05-2

Acetylcholinesterase promotes apoptosis in insect neurons

Apoptosis plays a major role in development, tissue renewal and the progression of degenerative diseases. Studies on various types of mammalian cells reported a pro-apoptotic function of acetylcholinesterase (AChE), particularly in the formation of the apoptosome and the degradation of nuclear DNA. While three AChE splice variants are present in mammals, invertebrates typically express two ache genes that code for a synaptically located protein and a protein with non-synaptic functions respectively. In order to investigate a potential contribution of AChE to apoptosis in insects, we selected the migratory locust Locusta migratoria. We established primary neuronal cultures of locust brains and characterized apoptosis progression in vitro. Dying neurons displayed typical characteristics of apoptosis, including caspase-activation, nuclear condensation and DNA fragmentation visualized by TUNEL staining. Addition of the AChE inhibitors neostigmine and territrem B reduced apoptotic cell death under normal culture conditions. Moreover, both inhibitors completely suppressed hypoxia-induced neuronal cell death. Exposure of live animals to severe hypoxia moderately increased the expression of ace-1 in locust brains in vivo. Our results indicate a previously unreported role of AChE in insect apoptosis that parallels the pro-apoptotic role in mammalian cells. This similarity adds to the list of apoptotic mechanisms shared by mammals and insects, supporting the hypothesized existence of an ancient, complex apoptosis regulatory network present in common ancestors of vertebrates and insects

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP